Against the backdrop of global warming and exhaustion of fossil resources, production of chemical products using renewable resources has attracted attention as an emerging industry for realizing a low-carbon society.
4-HBA is a useful substance used as a raw material for liquid-crystal polymers, as a raw material for the synthesis of paraben, which is an antimicrobial agent, and the like.
Currently, 4-HBA is produced by chemical conversion from crude oil as a raw material. Examples of chemical 4-HBA production processes include a process in which phenol, potassium hydroxide, and carbon dioxide are reacted under high-pressure conditions.
Such a process depends on fossil materials for phenol as the starting material, and in addition, places a great burden on the environment because the process requires strong alkali, carbon dioxide, and high-temperature and high-pressure conditions, and produces hazardous liquid waste.
Therefore, there is a strong need to establish an energy-saving, environment-conscious process that allows biological production of and produces a reduced amount of hazardous liquid waste.
However, biological production of 4-HBA from renewable resources is less productive as compared to production of lactic acid or ethanol because the metabolic reaction from a raw material sugar consists of a great many steps. In addition, there are problems, such as inhibition of bacterial growth by produced 4-HBA and cytotoxicity of 4-HBA. Therefore, industrial production of 4-HBA has not been achieved.
Using Escherichia coli, it has been revealed that 4-HBA is synthesized from chorismic acid, which is an intermediate in the shikimate pathway involved in the synthesis of aromatic amino acids etc., by chorismate-pyruvate lyase encoded by ubiC (Non Patent Literature 1 and 2, Patent Literature 1 and 2).
There is a report of introduction of a chorismate pyruvate-lyase gene (ubiC) of Escherichia coli into a different kind of microorganism, Klebsiella pneumoniae, as a host, in an attempt to produce 4-HBA (Hon Patent Literature 3). Also, there is a report of fermentative production of 4-HBA in an Escherichia coli in which the shikimic acid pathway is reinforced (Non Patent Literature 4). In an attempt to avoid the growth inhibition or the toxic action by 4-HBA, there are reports of selection of 4-HBA-resistant strains and of culture in the presence of an ion-exchange resin, but practically sufficient 4-HBA productivity has not been achieved (Non Patent Literature 2).
Regarding ubiCs of other living organisms than Escherichia coli, the ubiC of Rhodobactec sphaeroides has been reported. However, an Escherichia coli transformant highly expressing ubiC and a Rhodobacter sphaeroides transformant highly expressing ubiC are capable of producing 4-HBA only at low concentrations, which are not practically sufficient (Patent Literature 3). Also, despite the description that the ubiC of Rhodobacter sphaeroides can complement the ubiC of Escherichia coli in a disruptant of Escherichia coli lacking the ubiC gene, the literature does not include any enzymatic activity values, description regarding enzymatic characteristics, or detailed description regarding comparison with enzymes from other living organisms.
The UbiC of Escherichia coli has already been enzymatically analyzed in detail, and is known to be strongly inhibited by the product, 4-HBA (product inhibition) (Non Patent Literature 2 and 5). Therefore, in order to establish a high 4-HBA-producing strain aiming at a higher production of 4-HBA, obtaining a highly active ubiC and obtaining a resistant ubiC against product inhibition by 4-KBA are extremely important.